A tunable interleukin-18 (IL-18) platform engineered for complete escape from the decoy receptor IL-18 binding protein (IL-18BP)

Background: IL-18 is a potent immune cytokine that promotes CD8⁺ T-cell- and NK-cell-mediated antitumor responses. Its therapeutic potential is limited by rapid neutralization by IL-18BP, poor stability, short half-life, and systemic toxicity. Multiple efforts have attempted to generate IL-18 variants that escape IL-18BP neutralization, but current variants either retain residual IL-18BP binding or require extensive mutations. Our goal was to produce IL-18 variants fully resistant to IL-18BP with minimal mutations while maintaining controlled IL-18 activity via engaging the IL-18 receptor (IL-18R1). Methods: Using structural modeling with our AI-driven Intelligent Biomolecular Discovery Platform, we identified amino acids critical for IL-18BP and IL-18R1 interactions. A combinatorial IL-18 library was constructed by site-directed mutagenesis and displayed on yeast. Variants that lost IL-18BP binding but retained IL-18R1 interaction were enriched by FACS. DNA coding sequences of positive clones were Sanger sequenced, subcloned into mammalian expression vector. And recombinant proteins were purified. IL-18R1-specific binding and IL-18BP escape were confirmed by ELISA, and binding KDs were determined by bio-layer interferometry. Functional activity was assessed by IFN-gamma production from human PBMCs in the presence of 5 µg/mL IL-18BP. Results: We initially identified a positive clone named E8 which showed reducing binding affinity to IL-18BP. We further obtained more than 200 clones which completely lost binding to IL-18BP from a secondary library based on the sequence of E8. Multiple sequence alignment revealed that clones with few amino acid mutations could fully abolish IL-18BP binding while retaining minimal IL-18R1 interaction. In addition, specific amino acid positions were identified whose single or combined mutation restored IL-18R1 affinity. Combinatorial mutations of specificity-related and affinity-related amino acids yielded a panel of over 100 IL-18 variants with IL-18R1 binding KDs ranging from 10⁻⁹ to 10⁻⁷ M and complete loss of IL-18BP binding. Representative variants activated human PBMCs with graded IFN-gamma responses despite presence of high IL-18BP levels. One representative variant was fused to a anti-PD-1 IgG and demonstrated a tumor growth inhibition of 98% in mouse MC38 tumor model. Conclusion: We developed a series of engineered IL-18 variants (IL-18v) that fully escape IL-18BP neutralization while providing tunable IL-18R1 affinity. This IL-18v platform enables customizable cytokine potency and supports diverse research and therapeutic applications, including incorporation into fusion proteins, targeted cytokine therapies, and next-generation immunotherapy strategies.